| May
1, 2005 |
| By:
Haiying
Grunenwald, Judith
E. Meis, Katharine
Kramer |
| Pharmaceutical
Discovery |
|
Real-time RT-PCR is used extensively to quantify and validate gene
expression analysis. PCR efficiency is a critical parameter for accurate
quantification of gene expression, which can be optimized easily with
FailSafe™ Real-Time PCR Optimization Kits.
Introduction Real-time reverse
transcriptase polymerase chain reaction (RT-PCR) is a rapid
quantification technique that provides high sensitivity, good
reproducibility and a wide dynamic range (1, 2). Relative quantification
methods, based on mathematical algorithms, are being used to compare the
expression levels of a target gene to a standardized, constitutive
reference gene (1, 3). A factor in this equation is the PCR efficiency,
which is unique to each primer pair and has a major impact on the
accuracy of the calculated expression ratios (1).
Accurate relative real-time PCR requires careful primer design,
optimized reagents (template quality and quantity, buffer composition),
optimized cycling conditions (annealing temperatures) and similar
optimized PCR efficiencies for both the target gene and the reference
gene (4). Here we describe a convenient way to quickly optimize
real-time PCR buffer conditions, which leads to optimized PCR
efficiencies, using FailSafe™ Real-Time PCR Optimization Kits.
FailSafe™ Kits are designed for either fluorescent probe or SYBR®
Green I Dye detection.
Experimental Conditions The
FailSafe™ PROBES Real-Time PCR Optimization Kit uses eight unique
PreMixes, which contain a range of reagent concentrations and include
dNTPs, buffer, salts and EPICENTRE's (Madison, WI, USA) PCR Enhancer
with Betaine. The kit includes the FailSafe™ PCR Enzyme Blend, which
provides optimal sensitivity, specificity and PCR efficiency for both
single amplification and multiplex real-time PCR.
To select optimum amplification conditions for the human GAPDH gene,
HeLa cDNA was prepared from 10 pg of total RNA using EPICENTRE's
thermostable MonsterScript™ Reverse Transcriptase in a 20-μL
reaction. Eight (one with each PreMix) 25-μL reactions were set up
using 1 μL of the prepared cDNA, 12.5 pmol of each GAPDH primer and
100 nmol of a FAM/BHQ1 dual-labeled probe.
Figure 1. An amplification plot of
the human GAPDH gene from HeLa cDNA in each of the eight
FailSafe™ PROBES Real-Time PCR PreMixes.
|
Results Based
on the threshold cycle and amplification plot of the reactions (Figure
1), PreMix 3 was determined to provide optimum PCR conditions for this
template, primer and probe combination.
Figure 2. An amplification plot of
3 × 102 to 3 × 108 copies of a plasmid
containing the human GAPDH gene and the resulting standard
curve.
|
Subsequent PCR reactions used PreMix
3, the same primers and probe and from 3 × 102 to 3 × 108
copies of a plasmid containing the human GAPDH gene, in duplicate. The
standard curve calculated from the reactions has a correlation
coefficient of 1.0 and gives a PCR efficiency of 100.6% (Figure 2).
Acceptable PCR efficiencies range from 90% to 110%.
Conclusions Optimizing
PCR efficiency for GAPDH, a common housekeeping gene, took only one
real-time PCR assay using the eight FailSafe™ PROBE PreMixes. To
easily optimize real-time PCR with SYBR® Green I Dye
detection, FailSafe™ GREEN Real-Time PCR Optimization Kits are
available for both tube and capillary formats. These kits contain 12
(tube) or eight (capillary) unique PreMixes and the FailSafe™ PCR
Enzyme Blend.
References
1. M.W. Pfaffl et al., Nucleic Acids Res. 30, e36 (2002).
2. S.N. Peirson et al., Nucleic
Acids Res. 31, e73 (2003).
3. M.S. Rajeevan et al., J. Mol.
Diagn. 3, 26-31 (2001).
4. A. Giulietti et al., Methods 25,
386-401 (2001).
EPICENTRE Biotechnologies
726 Post Rd., Madison, WI 53713-4620 USA
Tel. 800-284-8474 or 608-258-3080
Fax 608-258-3088
techhelp@EpiBio.com
www.EpiBio.com
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